Biopolymer arrays and their fabrication

ABSTRACT

A method of fabricating an addressable array of biopolymers on a substrate using a biomonomer with a first linking group which must be activated for linking to a substrate bound moiety, and apparatus and computer program products for executing the method. The method includes forming on a region of the substrate carrying the substrate bound moiety, a solid activator composition. A biomonomer containing fluid composition is deposited on the region so that the solid activator activates the first linking group and the biomonomer links to the substrate bound moiety. The foregoing steps may be repeated, wherein a biomonomer deposited and linked to the substrate bound moiety in one cycle is the substrate bound moiety for the next cycle, so as to form the biopolymer.

FIELD OF THE INVENTION

This invention relates to arrays, particularly polynucleotide arrayssuch as DNA arrays, which are useful in diagnostic, screening, geneexpression analysis, and other applications.

BACKGROUND OF THE INVENTION

Polynucleotide arrays (such as DNA or RNA arrays), are known and areused, for example, as diagnostic or screening tools. Such arrays includeregions (sometimes referenced as features or spots) of usually differentsequence polynucleotides arranged in a predetermined configuration on asubstrate. The arrays, when exposed to a sample, will exhibit a bindingpattern. This binding pattern can be observed, for example, by labelingall polynucleotide targets (for example, DNA) in the sample with asuitable label (such as a fluorescent compound), and accuratelyobserving the fluorescence pattern on the array. Assuming that thedifferent sequence polynucleotides were correctly deposited inaccordance with the predetermined configuration, then the observedbinding pattern will be indicative of the presence and/or concentrationof one or more polynucleotide components of the sample.

Biopolymer arrays can be fabricated using either deposition of thepreviously obtained biopolymers or in situ synthesis methods. Thedeposition methods basically involve depositing biopolymers atpredetermined locations on a substrate which are suitably activated suchthat the biopolymers can link thereto. Biopolymers of different sequencemay be deposited at different regions of the substrate to yield thecompleted array. Typical procedures known in the art for deposition ofpreviously obtained polynucleotides, particularly DNA such as wholeoligomers or cDNA, are to load a small volume of DNA in solution in oneor more drop dispensers such as the tip of a pin or in an open capillaryand, touch the pin or capillary to the surface of the substrate. Such aprocedure is described in U.S. Pat. No. 5,807,522. When the fluidtouches the surface, some of the fluid is transferred. The pin orcapillary must be washed prior to picking up the next type of DNA forspotting onto the array. This process is repeated for many differentsequences and, eventually, the desired array is formed. Alternatively,the DNA can be loaded into a drop dispenser in the form of an inkjethead and fired onto the substrate. Such a technique has been described,for example, in PCT publications WO 95/25116 and WO 98/41531, andelsewhere.

The in situ synthesis methods include those described in U.S. Pat. No.5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 andthe references cited therein for synthesizing polynucleotides(specifically, DNA) using phosphoramidite or other chemistry. Such insitu synthesis methods can be basically regarded as iterating thesequence of depositing droplets of: (a) a protected monomer ontopredetermined locations on a substrate to link with either a suitablyactivated substrate surface (or with a previously deposited deprotectedmonomer); (b) deprotecting the deposited monomer so that it can nowreact with a subsequently deposited protected monomer; and (c)depositing another protected monomer for linking. Different monomers maybe deposited at different regions on the substrate during any one cycleso that the different regions of the completed array will carry thedifferent biopolymer sequences as desired in the completed array. One ormore intermediate further steps may be required in each iteration, suchas oxidation and washing steps.

It is important in such arrays that features actually be present, thatthey are put down accurately in the desired pattern, are of the correctsize, and that the DNA is uniformly coated within the feature. However,in the conventional in situ methods for polynucleotide arrays,phosphoramidite nucleoside monomers are used. In order for thephosphoramidite group to link to a hydroxyl of a previously depositeddeprotected polynucleotide monomer, it must first be activated usuallyby using a weak acid such as tetrazole. However, an activatedphosphoramidite is highly reactive with moisture in the air. This leadsto a reduction in deposited monomer available for reaction. Furthermore,since water tends to be adsorbed initially at the surface of a dropletwhich is being used in one cycle of forming a polynucleotide at afeature, the phosphoramidite concentration at the surface of the dropletwill tend to be lowest. Consequently, the concentration of a completedprobe polynucleotide at a feature of the array, tends to decrease fromthe center of a feature toward its perimeter. This leads to a decreasein the total signal that should be available when a target to which thatpolynucleotide hybridizes, is detected. Furthermore, since water vaporconcentration the ambient atmosphere may vary, such signal may also varyfrom array to array, leading to inconsistency in absolute signalgenerated from different arrays of a batch when the same concentrationof a target is encountered. The foregoing problems particularly existwhere the phosphoramidite is mixed with activator and the mixturedeposited as a droplet on the substrate, such as described in PCTpublication WO98-41531. The foregoing reference also states that theactivator can be deposited onto a previously deposited dropletcontaining the phosphoramidite. However, the potential for the activatedphorphoramidite to react with moisture in the ambient atmosphere stillexists. Furthermore, when one droplet is deposited on the other, thereis no guarantee of efficient mixing such that the activatedphosphoramidite will be evenly present at the substrate surface.

It would be desirable then, in the fabrication of arrays of biopolymersusing biomonomers with a linking group which must be activated (such asa phosphoramidite), to provide a means by which potential reactivity ofthe activated biomonomer with an ambient atmosphere component (such aswater vapor in air) can be kept low.

SUMMARY OF THE INVENTION

The present invention then, provides a method of fabricating anaddressable array of biopolymers on a substrate using a biomonomer witha first linking group which must be activated for linking to a substratebound moiety. The method includes forming on a region of the substratecarrying the substrate bound moiety, a solid activator composition. Abiomonomer containing fluid composition is deposited on the region sothat the solid activator activates the first linking group and thebiomonomer links to the substrate bound moiety. Typically, the foregoingsteps are repeated, with a biomonomer deposited and linked to thesubstrate bound moiety in one cycle acting as the substrate bound moietyfor the next cycle, so as to form the biopolymer. However, it will beappreciated that one or more such cycles can be performed.

In the fabrication of a typical array with multiple features, all of theforegoing steps are repeated at each of multiple different regions onthe same substrate, where it is desired to form the features. Generally,the biomonomer containing fluid will be deposited after forming thesolid activator composition on the region or regions. As to of formingthe solid activator composition at the region, one way of accomplishingthis is to deposit a composition of solid activator as a fluidcomposition, and allowing fluid to evaporate. In this case, the fluidcomposition may have less than 20% by weight of solid activator content,for example 3% to 20% by weight (or even less than 10% by weight). Inone aspect of the method, a sufficient amount of the biomonomer fluidcomposition may be deposited at a region so as to cover an area greaterthan that covered by the solid activator composition at the same region(or greater than the area covered by the activator fluid composition atthe same region, when the solid activator is deposited as a fluidcomposition).

The biopolymers may in particular be polynucleotides (for example, DNA),in which case the biomonomer is a nucleoside monomeric unit. Theactivated biomonomer may particularly react with a component in ambientatmosphere. For example, where the biomonomer is a phosphoramidite, itis reactive with water vapor in air. The same or different biomonomerscan be deposited at the same region in different cycles. Furthermore,the same or different fluids can be used for the biomonomer fluid andactivator fluid compositions. In one particular case, the fluid of thesolid activator fluid composition may have a boiling point of less than100° C. (or less than 90° C. or even less than 85° C.) while the fluidof the biomonomer containing fluid composition has a boiling point ofgreater than 100° C. (or greater than 105° C. or even greater than 110°C.).

In a particular aspect of the above array fabrication methods of thepresent invention, a deposition system is used which has a head withmultiple pulse jets each of which can dispense fluid droplets onto thesubstrate. Each such jet includes a chamber with an orifice, and anejector which, when activated, causes a droplet to be ejected from theorifice. Such deposition apparatus can be used to dispense droplets ofthe biomonomer containing fluid and, optionally, to also dispensedroplets of the solid activator fluid composition. Alternatively, itwill be appreciated that in methods of the present invention forfabricating arrays, the fluid composition of solid activator could beapplied as a continuous layer over multiple regions.

The present invention also provides a method of evaluating for thepresence of a target polynucleotide in a sample, using an addressablearray fabricated in accordance with any of the methods of the presentinvention. The evaluation method comprises exposing the sample to thearray, such that target polynucleotide which may be present will bind toone or more predetermined regions of the array. Optionally, a bindingpattern on the array may then be observed and the presence of the targetpolynucleotide evaluated based on the observed binding pattern. However,this can be done either within a short time following the foregoingsteps, or potentially at some indefinite later time.

The present invention further provides an apparatus for fabricating anaddressable array of biopolymers on a substrate according to a targetpattern. The apparatus includes a deposition system which can separatelydispense onto a substrate, fluid compositions of different biomonomerseach with a first linking group which must be activated for linking to asubstrate bound moiety, and a fluid composition of a solid activator.The apparatus further includes a processor to operate the depositionsystem. The processor derives from the target array pattern a targetdrive pattern for operating the deposition system to form the array. Thetarget drive pattern includes instructions to the deposition system todeposit the fluid composition of solid activator at each region at whicha biomonomer is to be deposited, separate from and preceding depositionof the biomonomer. The deposition system can include one or multiplepulse jets which can dispense droplets of the different biomonomer fluidcompositions, and at least one pulse jet which can separately dispensethe activator fluid composition. Each jet includes a chamber with anorifice, and an ejector which, when activated, causes a droplet to beejected from the orifice. Optionally, the target drive pattern mayinclude ejector instructions such that a droplet of biomonomer fluidcomposition deposited at a region will cover an area greater than thatcovered by a preceding droplet of activator fluid composition at thesame region.

A still further aspect of the present invention includes a computerprogram product, for use on an apparatus (such as an apparatus of theabove described type) for fabricating an addressable array of biopolymerprobes on a substrate according to a target array pattern. The programproduct includes a computer readable storage medium having a computerprogram stored thereon. The program, when loaded into a computer of theapparatus derives from the target array pattern a target drive patternfor operating a deposition system of the apparatus to form the array.This target drive pattern includes instructions to the deposition systemto deposit the fluid composition of solid activator at each region atwhich a biomonomer monomer is to be deposited, separate from andpreceding deposition of the biomonomer. Optionally, the target drivepattern may include instructions to the deposition system to depositsufficient biomonomer fluid composition at a region which will cover anarea greater than that covered by a preceding droplet of activator fluidcomposition at the same region.

While the substrates referenced in the aspects of the apparatus, methodsand programs of the present invention described above, carrybiopolymers, the present invention contemplates that these particularmoieties can readily be replaced with other moieties (such as otherchemical or biochemical moieties, for example various small molecules)in any of the apparatus, methods or kits of the present invention, whereactivation of a component is required and particularly where theactivated component is more reactive with a component of the ambientatmosphere than is the unactivated component. Thus, wherever a referenceis made to biopolymers, this can be replaced with a reference to anysuch moieties.

The present invention then, including methods, apparatus, and computerprogram products thereof, can provide any one or more, of a number ofuseful benefits. For example, in the fabrication of arrays ofbiopolymers using biomonomers with a linking group which must beactivated (such as a phosphoramidite), the present invention provides ameans by which potential reactivity of the activated biomonomer with anambient atmosphere component can be kept low.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a substrate bearing multiple arrays, asmay be produced by a method and apparatus of the present invention;

FIG. 2 is an enlarged view of a portion of FIG. 1 showing some of theidentifiable individual regions (or “features”) of a single array ofFIG. 1;

FIG. 3 is an enlarged cross-section of a portion of FIG. 2;

FIG. 4 is an enlarged cross-section illustrating a sequence of events ina method of the present invention during formation of one feature of anarray;

FIG. 5 is an enlarged view of a portion of FIG. 4; and

FIG. 6 is a schematic view of apparatus of the present invention; and

FIG. 7 is a view similar to that of FIG. 2 but illustrating a preferredmethod in which the area occupied by biomonomer solution at a regionduring any one cycle, is greater than that covered by a deposited solidactivator layer previously deposited in the same cycle;

To facilitate understanding, identical reference numerals have beenused, where practical, to designate identical elements that are commonto the figures.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In the present application, unless a contrary intention appears, thefollowing terms refer to the indicated characteristics. A “biopolymer”is a polymer of one or more types of repeating units. Biopolymers arefound in biological systems and particularly include peptides orpolynucleotides, as well as such compounds composed of or containingamino acid or nucleotide analogs or non-nucleotide groups. This includespolynucleotides in which the conventional backbone has been replacedwith a non-naturally occurring or synthetic backbone, and nucleic acidsin which one or more of the conventional bases has been replaced with asynthetic base capable of participating in Watson-Crick type hydrogenbonding interactions. Polynucleotides include single or multiplestranded configurations, where one or more of the strands may or may notbe completely aligned with another. A “nucleotide” refers to a subunitof a nucleic acid and has a phosphate group, a 5 carbon sugar and anitrogen containing base, as well as analogs of such subunits. A“nucleoside” is of the same structure but without a phosphate group.Specifically, a “biopolymer” includes DNA (including cDNA), RNA andoligonucleotides. An “oligonucleotide” generally refers to a nucleotidemultimer of about 10 to 100 nucleotides in length, while a“polynucleotide” includes a nucleotide multimer having any number ofnucleotides. A “biomonomer” references a single unit, which can belinked with the same or other biomonomers to form a biopolymer (forexample, a single amino acid or nucleotide with two linking groups oneor both of which may have removable protecting groups). A biomonomerfluid or biopolymer fluid reference a liquid containing either abiomonomer or biopolymer, respectively (typically in solution). A“phosphoramidite” includes a group of the structure of formula (I)below:

wherein either X is a linking atom such as O or S and may be the same ordifferent; Y is a protecting group such as cyanoethyl; Z may be ahalogen (particularly Cl or Br) or a secondary amino group such asmorpholino or N(lower alkyl)₂ where the alkyl groups are the same ordifferent, preferably N(i-propyl)₂. By “lower alkyl” is referenced 1 to8 C atoms. A nucleoside phosphoramidite has a nucleoside or a nucleosideanalog with the sugar ring bonded to the free bond on the X in formula(I). For example, one particular nucleoside phosphoramidite isrepresented by formula (II) below:

wherein B is a nucleoside base, and DMT is dimethoxytrityl. The O (whichmay instead be replaced by S) to which DMT is bonded, acts as a secondlinking group which is protected by the DMT. Protecting groups otherthan DMT may be used, and their removal during deprotection is known inoligonucleotide synthesis. Other nucleoside phosphoramidites are alsoknown, for example ones in which the phosphoramidite group is bonded toa different location on the 5-membered sugar ring. Phosphoramidites andnucleoside phosphoramidites are described in U.S. Pat. No. 5,902,878,U.S. Pat. No. 5,700,919, U.S. Pat. No. 4,415,732, PCT publication WO98/41531 and the references cited therein, among others. A “group”includes both substituted and unsubstituted forms. An “addressablearray” includes any one or two dimensional arrangement of discreteregions (or “features”) bearing particular biopolymer moieties (forexample, different polynucleotide sequences) associated with that regionand positioned at particular predetermined locations on the substrate(each such location being an “address”). These regions may or may not beseparated by intervening spaces. It will also be appreciated thatthroughout the present application, words such as “upper”, “lower” andthe like are used with reference to a particular orientation of theapparatus with respect to gravity, but it will be understood that otheroperating orientations of the apparatus or any of its components, withrespect to gravity, are possible. Reference to a “droplet” beingdispensed from a pulse jet herein, merely refers to a discrete smallquantity of fluid (usually less than about 1000 pL) being dispensed upona single pulse of the pulse jet (corresponding to a single activation ofan ejector) and does not require any particular shape of this discretequantity. When a “spot” is referred to, this may reference a dried spoton the substrate resulting from drying of a dispensed droplet, or a wetspot on the substrate resulting from a dispensed droplet which has notyet dried, depending upon the context. “Fluid” is used herein toreference a liquid. Use of the singular in reference to an item,includes the possibility that there may be multiple numbers of thatitem. A “solid” may still have some amount of a carrier fluid, such as asolvent, present. However, typically a “solid” will have no more than20% by weight (and often less than 10% or 5%, or 1%, by weight, of suchcarrier fluid present). A “solid activator” is one which is solid at theoperating temperature at which it is used (normally at around a typicalroom temperatures, such as between 10° C. to 30° C.). By one item being“remote” from another is referenced that they are at least in differentbuildings, and may be at least one, at least ten, or at least onehundred miles apart.

Referring first to FIGS. 1-3, typically the present invention willproduce multiple identical arrays 12 (only some of which are shown inFIG. 1) across a complete front surface 11 a of a single substrate 10(which also has a back surface 11 b). However, the arrays 12 produced ona given substrate need not be identical and some or all could bedifferent. Each array 12 will contain multiple spots or features 16. Thearrays 12 are shown as being separated by spaces 13. A typical array 12may contain from 100 to 100,000 features. All of the features 16 may bedifferent, or some or all could be the same. Each feature carries apredetermined polynucleotide having a particular sequence, or apredetermined mixture of polynucleotides. This is illustratedschematically in FIG. 3 where different regions 16 are shown as carryingdifferent polynucleotide sequences. While arrays 12 are shown separatedfrom one another by spaces 13, and the features 16 are separated fromone another by spaces, such spaces in either instance are not essential.

Referring to FIGS. 4 and 5 in particular, the principle of the presentinvention can be understood. These FIGS. are not to scale, with somefeatures being exaggerated for clarity. It will be assumed that asubstrate bound moiety is present at least at the location of eachfeature or region to be formed. Such substrate bound moiety may, forexample, be a nucleoside monomer has been deposited and deprotected atthe location of each feature, such that the second linking group isavailable for linking to another activated nucleoside monomer.Alternatively, the substrate bound moiety may be a suitable linkinggroup previously attached to substrate 10. Both of these steps are knownin in situ fabrication techniques. FIGS. 4 and 5 illustrate only oneregion of an array being fabricated. A droplet 40 of a solution of asolid activator is deposited onto the region carrying the substratebound moiety. The solvent of the solution is allowed to evaporate toform a layer 42 of solid activator on substrate 10. Given the volume ofa typical droplet, and the solvents which may be used, as discussedherein, this evaporation will typically take place in less than onesecond (and may be completed in less than 0.5 or even 0.25 seconds). Adroplet 44 of a biomonomer solution, such as a nucleosidephosphoramidite monomer, is then deposited onto the region. Preferably,but not necessarily, this droplet 44 will cover an area which is greaterthan that covered by the droplet 40 and hence greater than the areacovered by layer 42. As a result of the above steps, solid activator oflayer 42 will activate the first linking group of the biomonomer,specifically the phosphoramidite group of a nucleoside monomer, suchthat the activated group will then link with the substrate bound moiety(again, a linking group previously attached to substrate 10 or adeprotected nucleoside monomer deposited in a previous cycle).

The above steps can be repeated at the illustrated region in FIGS. 4 and5, until the desired biopolymer has been synthesized. It will beunderstood however, that intermediate oxidation, deprotection, washingand other steps may be required between cycles, as is well known in theart of synthesizing biopolymers (such as oligonucleotides). These cyclesmay be repeated using different or the same biomonomers, at multipleregions over multiple cycles, as required to fabricate the desired arrayor arrays 12 on substrate 10.

As discussed above, the activated biomonomer (such as the activatedphosphoramidite) may be reactive with a component in the ambientatmosphere (such as water vapor). However, as best seen in FIG. 5,activated biomonomer is generated adjacent the layer 42 of solidactivator. Even if the ambient atmosphere component of concern maydissolve in droplet 44 to some extent, possibly even forming a frontalboundary 48 therein, it will be appreciated that activated monomer nearlayer 42 will not be exposed to that component for some time, if at all.This will allow time for the activated monomer to link to the surfacebound moiety.

Referring now to FIG. 6 the apparatus shown includes a substrate station20 on which can be mounted a substrate 10. Pins or similar means (notshown) can be provided on substrate station 20 by which to approximatelyalign substrate 10 to a nominal position thereon. Substrate station 20can include a vacuum chuck connected to a suitable vacuum source (notshown) to retain a substrate 14 without exerting too much pressurethereon, since substrate 14 is often made of glass. A flood station 68is provided which can expose the entire surface of substrate 10, whenposition beneath station 68 as illustrated in broken lines in FIG. 4, toa fluid typically used in the in situ process, and to which all featuresmust be exposed during each cycle (for example, oxidizer, deprotectionagent, and wash buffer).

A dispensing head 210 is retained by a head retainer 208. Thepositioning system includes a carriage 62 connected to a firsttransporter 60 controlled by processor 140 through line 66, and a secondtransporter 100 controlled by processor 140 through line 106.Transporter 60 and carriage 62 are used execute one axis positioning ofstation 20 (and hence mounted substrate 10) facing the dispensing head210, by moving it in the direction of arrow 63, while transporter 100 isused to provide adjustment of the position of head retainer 208 (andhence head 210) in a direction of axis 204. In this manner, head 210 canbe scanned line by line, by scanning along a line over substrate 10 inthe direction of axis 204 using transporter 100, while line by linemovement of substrate 10 in a direction of axis 63 is provided bytransporter 60. Transporter 60 can also move substrate holder 20 toposition substrate 10 beneath flood station 68 (as illustrated in brokenlines in FIG. 4). Head 210 may also optionally be moved in a verticaldirection 202, by another suitable transporter (not shown). It will beappreciated that other scanning configurations could be used. It willalso be appreciated that both transporters 60 and 100, or either one ofthem, with suitable construction, could be used to perform the foregoingscanning of head 210 with respect to substrate 10. Thus, when thepresent application recites “positioning” one element (such as head 210)in relation to another element (such as one of the stations 20 orsubstrate 10) it will be understood that any required moving can beaccomplished by moving either element or a combination of both of them.The head 210, the positioning system, and processor 140 together act asthe deposition system of the apparatus. An encoder 30 communicates withprocessor 140 to provide data on the exact location of substrate station20 (and hence substrate 10 if positioned correctly on substrate station20), while encoder 34 provides data on the exact location of holder 208(and hence head 210 if positioned correctly on holder 208). Any suitableencoder, such as an optical encoder, may be used which provides data onlinear position.

Head 210 may be of a type commonly used in an ink jet type of printerand may, for example, include five or more chambers (at least one foreach of four nucleoside phosphoramidite monomers plus at least one for asolution of solid activator) each communicating with a corresponding setof multiple drop dispensing orifices and multiple ejectors which arepositioned in the chambers opposite respective orifices. Each ejector isin the form of an electrical resistor operating as a heating elementunder control of processor 140 (although piezoelectric elements could beused instead). Each orifice with its associated ejector and portion ofthe chamber, defines a corresponding pulse jet. It will be appreciatedthat head 210 could, for example, have more or less pulse jets asdesired (for example, at least ten or at least one hundred pulse jets).Application of a single electric pulse to an ejector will cause adroplet to be dispensed from a corresponding orifice. Certain elementsof the head 210 can be adapted from parts of a commercially availablethermal inkjet print head device available from Hewlett-Packard Co. aspart no. HP51645A. Alternatively, multiple heads could be used insteadof a single head 210, each being similar in construction to head 210 andbeing provided with respective transporters under control of processor140 for independent movement. In this alternate configuration, each headmay dispense a corresponding biomonomer (for example, one of fournucleoside phosphoramidites) or a solution of a solid activator.

As is well known in the ink jet print art, the amount of fluid that isexpelled in a single activation event of a pulse jet, can be controlledby changing one or more of a number of parameters, including the orificediameter, the orifice length (thickness of the orifice member at theorifice), the size of the deposition chamber, and the size of theheating element, among others. The amount of fluid that is expelledduring a single activation event is generally in the range about 0.1 to1000 pL, usually about 0.5 to 500 pL and more usually about 1.0 to 250pL. A typical velocity at which the fluid is expelled from the chamberis more than about 1 m/s, usually more than about 10 m/s, and may be asgreat as about 20 m/s or greater. As will be appreciated, if the orificeis in motion with respect to the receiving surface at the time anejector is activated, the actual site of deposition of the material willnot be the location that is at the moment of activation in aline-of-sight relation to the orifice, but will be a location that ispredictable for the given distances and velocities.

The apparatus can deposit droplets to provide features which may havewidths (that is, diameter, for a round spot) in the range from a minimumof about 10 μm to a maximum of about 1.0 cm. In embodiments where verysmall spot sizes or feature sizes are desired, material can be depositedaccording to the invention in small spots whose width is in the rangeabout 1.0 μm to 1.0 mm, usually about 5.0 μm to 500 μm, and more usuallyabout 10 μm to 200 μm.

The apparatus further includes a display 310, speaker 314, and operatorinput device 312. Operator input device 312 may, for example, be akeyboard, mouse, or the like. Processor 140 has access to a memory 141,and controls print head 210 (specifically, the activation of theejectors therein), operation of the positioning system, operation ofeach jet in print head 210, and operation display 310 and speaker 314.Memory 141 may be any suitable device in which processor 140 can storeand retrieve data, such as magnetic, optical, or solid state storagedevices (including magnetic or optical disks or tape or RAM, or anyother suitable device, either fixed or portable). Processor 140 mayinclude a general purpose digital microprocessor suitably programmedfrom a computer readable medium carrying necessary program code, toexecute all of the steps required by the present invention, or anyhardware or software combination which will perform those or equivalentsteps. The programming can be provided remotely to processor 141, orpreviously saved in a computer program product such as memory 141 orsome other portable or fixed computer readable storage medium using anyof those devices mentioned below in connection with memory 141. Forexample, a magnetic or optical disk 324 may carry the programming, andcan be read by disk reader 326.

Operation of the apparatus of FIG. 4 in accordance with a method of thepresent invention, will now be described with reference to FIG. 6 inparticular. First, it will be assumed that memory 141 holds a targetdrive pattern. This target drive pattern is the instructions for drivingthe apparatus components as required to form the target array (whichincludes target locations and dimension for each spot) on substrate 10and includes, for example, movement commands to transporters 60 and 100as well as firing commands for each of the pulse jets in head 210co-ordinated with the movement of head 210 and substrate 10. This targetdrive pattern is based upon the target array pattern and can have eitherbeen input from an appropriate source (such as input device 312, aportable magnetic or optical medium, or from a remote server, any ofwhich communicate with processor 140), or may have been determined byprocessor 140 based upon an input target array pattern (using any of theappropriate sources previously mentioned) and the previously knownnominal operating parameters of the apparatus. The target drive patternfurther includes instructions to head 210 and the positioning system ofthe apparatus to deposit the solution of solid activator at each regionat which a biomonomer is to be deposited, separate from and precedingdeposition of the biomonomer. Further, it will be assumed that each offour chambers of head 210 has been loaded with four different nucleosidephosphoramidite monomers, while a fifth chamber has been loaded withactivating agent. It will also be assumed that flood station 68 has beenloaded with all necessary solutions. Operation of the followingsequences are controlled by processor 140, following initial operatoractivation, unless a contrary indication appears.

For any given substrate 10, the operation is basically as follows,assuming in situ preparation of a typical oligonucleotide using standardnucleoside phosphoramidite monomers as the biomonomers. A substrate 10is loaded onto substrate station 20 either manually by an operator, oroptionally by a suitable automated driver (not shown) controlled, forexample, by processor 140. A target drive pattern necessary to obtain atarget array pattern, is determined by processor 140 (if not alreadyprovided), based on nominal operating parameters of the apparatus. Theapparatus is then operated as follows: (a) if not the first cycle,position substrate 10 at flood station 68 and for all regions of thearrays being formed, deprotected previously deposited and linkedbiomonomer on substrate 10 at flood station 68; (b) move substrate 10 toreceive droplets from head 210 and deposit droplets of solution of solidactivator from one or more pulse jets of head 210 onto each region inaccordance with the target drive pattern for each of multiple arrays 12;(c) allow sufficient time for activator solution to evaporate leavinglayer 42 of solid activator; (c) dispense appropriate next biomonomeronto each region such that the first linking group is activated by solidactivator and links to previously deposited deprotected biomonomer; (d)move substrate 10 back to flood station 68 for oxidation, capping, andwashing steps over entire substrate as required; and (e) repeatforegoing cycle for all the regions of all desired arrays 12 until thedesired arrays are completed (note that the biomonomer deposited andlinked to the substrate bound moiety in one cycle becomes the substratebound moiety for the next cycle). During each cycle, the relative areascovered by the solid activator and a biopolymer solution depositedimmediately following formation of the solid activator, are illustratedin FIG. 7 which is a view similar to that of FIG. 2. In FIG. 7, for eachregion the area occupied by a droplet of a biomonomer solution isindicated by a solid line circle, while the lesser area occupied by alayer 42 of solid activator formed from a droplet deposited immediatelypreceding the droplet of biomonomer solution, is illustrated by a brokenline circle. Note that the area of the final features will be equal tothat of the broken line circles.

Note that during the above operation, pressure within head 210 can becontrolled as described in co-pending patent applications “FABRICATINGBIOPOLYMER ARRAYS”, by Caren et al., Ser. No. 09/302,922, and“PREPARATION OF BIOPOLYMER ARRAYS” by A. Schleifer et al., Ser. No.09/302,899, both filed Apr. 30, 1999 and both assigned to the sameassignee as the present application, and the references cited therein.Those references and all other references cited in the presentapplication, are incorporated into this application by reference.Processor 140 can execute the control of pressure within head 210.

With regard to the actual deposition sequence of biomonomer or activatorsolution droplets, as already mentioned, in this sequence processor 140will operate the apparatus according to the target drive pattern, bycausing the positioning system to position head 210 facing substratestation 20, and particularly the mounted substrate 10, and with head 210at an appropriate distance from substrate 10. Processor 140 then causesthe positioning system to scan head 210 across substrate 14 line by line(or in some other desired pattern), while co-ordinating activation ofthe ejectors in head 210 so as to dispense droplets in accordance withthe target pattern. This can be continued until all arrays 12 to beformed on substrate 10 have been completed. The number of spots in anyone array 12 can, for example, be at least ten, at least one hundred, atleast one thousand, or even at least one hundred thousand.

At this point the droplet dispensing sequence is complete. In analternative to the above described embodiment, the activator solutioncan be deposited as a film over the entire substrate 10, rather thanbeing deposited as droplets only at the location of the desiredfeatures. This can save time, although activator solution will be wastedsince only activator at locations of subsequently deposited biomonomer(the desired feature locations) will be used, and the rest will bewashed off.

In the case of phosphoramidites, suitable activators are known andinclude tetrazole, S-ethyl tetrazole, dicyanoimidazole (“DCI”), orbenzimidazolium triflate. As to solvents for the activator, any lowboiling point solvent as already mentioned, could be used provided it isotherwise compatible with the chemistry being used. In the case ofphosphoramidites a non-protic low boiling point solvent could be used,for example, acetonitrile, dioxane, toluene, ethylacetate, acetone,tetrahydrofuran, and the like. Solvents for the biomonomers are alreadyknown, such as those solvents described in PCT publication WO 98/41531and the references cited therein.

Arrays fabricated by methods and apparatus of the present invention, canbe used to evaluate for the presence of one or more targetpolynucleotides in a known manner. Basically, this involves exposing thesample, normally as a fluid composition, to the array, such that targetpolynucleotide which may be present will bind to one or morepredetermined regions of the array. The binding pattern on the array maythen be observed by any method (such as by observing a fluorescencepattern), and the presence of the target evaluated based, in whole or inpart, on the observed binding pattern.

Modifications in the particular embodiments described above are, ofcourse, possible. For example, where a pattern of arrays is desired, anyof a variety of geometries may be constructed other than the organizedrows and columns of arrays 12 of FIG. 1. For example, arrays 12 can bearranged in a series of curvilinear rows across the substrate surface(for example, a series of concentric circles or semi-circles of spots),and the like. Similarly, the pattern of regions 16 may be varied fromthe organized rows and columns of spots in FIG. 2 to include, forexample, a series of curvilinear rows across the substrate surface (forexample, a series of concentric circles or semi-circles of spots), andthe like. Even irregular arrangements of the arrays or the regionswithin them can be used, at least when some means is provided such thatduring their use the locations of regions of particular characteristicscan be determined (for example, a map of the regions is provided to theend user with the array).

The present methods and apparatus may be used to deposit biopolymers orother moieties on surfaces of any of a variety of different substrates,including both flexible and rigid substrates. Preferred materialsprovide physical support for the deposited material and endure theconditions of the deposition process and of any subsequent treatment orhandling or processing that may be encountered in the use of theparticular array. The array substrate may take any of a variety ofconfigurations ranging from simple to complex. Thus, the substrate couldhave generally planar form, as for example a slide or plateconfiguration, such as a rectangular or square or disc. In manyembodiments, the substrate will be shaped generally as a rectangularsolid, having a length in the range about 4 mm to 200 mm, usually about4 mm to 150 mm, more usually about 4 mm to 125 mm; a width in the rangeabout 4 mm to 200 mm, usually about 4 mm to 120 mm and more usuallyabout 4 mm to 80 mm; and a thickness in the range about 0.01 mm to 5.0mm, usually from about 0.1 mm to 2 mm and more usually from about 0.2 to1 mm. However, larger substrates can be used, particularly when such arecut after fabrication into smaller size substrates carrying a smallertotal number of arrays 12. Substrates of other configurations andequivalent areas can be chosen. The configuration of the array may beselected according to manufacturing, handling, and use considerations.

The substrates may be fabricated from any of a variety of materials. Incertain embodiments, such as for example where production of bindingpair arrays for use in research and related applications is desired, thematerials from which the substrate may be fabricated should ideallyexhibit a low level of non-specific binding during hybridization events.In many situations, it will also be preferable to employ a material thatis transparent to visible and/or UV light. For flexible substrates,materials of interest include: nylon, both modified and unmodified,nitrocellulose, polypropylene, and the like, where a nylon membrane, aswell as derivatives thereof, may be particularly useful in thisembodiment. For rigid substrates, specific materials of interestinclude: glass; fused silica, silicon, plastics (for example,polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, andblends thereof, and the like); metals (for example, gold, platinum, andthe like).

The substrate surface onto which the polynucleotide compositions orother moieties is deposited may be smooth or substantially planar, orhave irregularities, such as depressions or elevations. The surface maybe modified with one or more different layers of compounds that serve tomodify the properties of the surface in a desirable manner. Suchmodification layers, when present, will generally range in thicknessfrom a monomolecular thickness to about 1 mm, usually from amonomolecular thickness to about 0.1 mm and more usually from amonomolecular thickness to about 0.001 mm. Modification layers ofinterest include: inorganic and organic layers such as metals, metaloxides, polymers, small organic molecules and the like. Polymeric layersof interest include layers of: peptides, proteins, polynucleic acids ormimetics thereof (for example, peptide nucleic acids and the like);polysaccharides, phospholipids, polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyethyleneamines, polyarylenesulfides, polysiloxanes, polyimides, polyacetates, and the like, wherethe polymers may be hetero- or homopolymeric, and may or may not haveseparate functional moieties attached thereto (for example, conjugated),

Various modifications to the embodiments of the particular embodimentsdescribed above are, of course, possible. Accordingly, the presentinvention is not limited to the particular embodiments described indetail above.

What is claimed is:
 1. A method of fabricating an addressable array ofbiopolymers on a substrate using a biomonomer with a first linking groupwhich must be activated for linking to a substrate bound moiety,comprising, at each of multiple different regions of the substrate: (a)forming on a region of the substrate carrying the substrate boundmoiety, a solid activator composition; (b) depositing a biomonomercontaining fluid composition on the region so that the solid activatoractivates the first linking group and the biomonomer links to thesubstrate bound moiety; and (c) repeating steps (a) and (b), wherein abiomonomer deposited and linked to the substrate bound moiety in onecycle is the substrate bound moiety for the next cycle, so as to formthe biopolymer; so as to form the addressable array; wherein thebiomonomer fluid composition deposited at a region covers an areagreater than that covered by the solid activator composition at the sameregion.
 2. A method according to claim 1 wherein step (a), the solidactivator composition is formed prior to depositing the biomonomercontaining fluid, by depositing a composition of solid activator as afluid composition, and allowing fluid to evaporate.
 3. A methodaccording to claim 2 wherein the biomonomer fluid composition depositedat a region covers an area greater than that covered by the activatorfluid composition at the same region.
 4. A method according to claim 2wherein the fluid composition has less than 20% by weight of solidactivator content.
 5. A method according to claim 2 wherein thebiopolymers are polynucleotides and the biomonomer is a nucleosidemonomer.
 6. A method according to claim 5 wherein the polynucleotide isa DNA.
 7. A method according to claim 1 wherein the activated biomonomerreacts with a component in an ambient atmosphere.
 8. A method accordingto claim 5 wherein the biomonomer is a phosphoramidite.
 9. A methodaccording to claim 1 wherein different biomonomers are deposited indifferent cycles.
 10. A method according to claim 1 wherein the samebiomonomers are deposited in different cycles.
 11. A method according toclaim 2 wherein the biomonomer fluid composition includes a fluiddifferent from that of the activator fluid composition.
 12. A method ofevaluating for the presence of a target polynucleotide in a sample,using an addressable array fabricated in accordance with the method ofclaim 1, the method comprising: (a) exposing the sample to the array,such that target polynucleotide which may be present will bind to one ormore predetermined regions of the array; and (b) observing a bindingpattern on the array and evaluating the presence of the targetpolynucleotide based on the observed binding pattern.
 13. A method offabricating an addressable array of biopolymers on a substrate usingbiomonomers each with a first linking group which must be activated forlinking to a substrate bound moiety, and a deposition system with a headhaving multiple pulse jets each of which can dispense droplets of afluid onto a substrate, each jet including a chamber with an orifice,and including an ejector which, when activated, causes a droplet to beejected from the orifice, the method comprising: (a) depositing onto aregion of the substrate carrying the substrate bound moiety, a fluidcomprising of a solid activator; (b) allowing fluid of the compositionto evaporate to form the solid activator on the region; (c) thendepositing from a pulse jet onto the region, a droplet of a biomonomercontaining fluid composition so that the solid activator activates thefirst linking group and the biomonomer links to the substrate boundmoiety; and (d) repeating steps (a) through (c) at each of multipleregions of the substrate, wherein at each region a biomonomer depositedand linked to the substrate bound moiety in one cycle is the substratebound moiety for the next cycle, so as to form the addressable array ofbiopolymers; wherein a droplet of biomonomer fluid composition depositedat a region will cover an area greater than that covered by a precedingdroplet of activator fluid composition at the same region.
 14. A methodaccording to claim 13 wherein the fluid composition of solid activatoris deposited as a droplet from a pulse jet.
 15. A method according toclaim 13 wherein the fluid composition of solid activator is applied asa continuous layer over multiple regions.
 16. A method according toclaim 14 wherein the fluid of the solid activator fluid composition hasa boiling point of less than 100° C.
 17. A method according to claim 13wherein the biomonomer containing fluid composition uses a fluiddifferent from that of the fluid composition of solid activator.
 18. Amethod according to claim 17 wherein the fluid of the solid activatorfluid composition has a boiling point of less than 100° C., while thefluid of the biomonomer containing fluid composition has a boiling pointof greater than 100° C.
 19. A method according to claim 13 wherein theactivator fluid composition has less than 20% by weight solid activatorcontent.
 20. A method according to claim 13 wherein the biopolymers arepolynucleotides and the biomonomer is a nucleoside monomer.
 21. A methodaccording to claim 13 wherein the polynucleotide is a DNA.
 22. A methodaccording to claim 13 wherein the activated biomonomer reacts with acomponent in an ambient atmosphere.
 23. A method according to claim 13wherein the biomonomer is a phosphoramidite.
 24. A method according toclaim 13 wherein, for each of the multiple regions, differentbiomonomers are deposited in different cycles.
 25. A method ofevaluating for the presence of a target polynucleotide in a sample,using an addressable array fabricated in accordance with the method ofclaim 13, the method comprising: (a) exposing the sample to the array,such that target polynucleotide which may be present will bind to one ormore predetermined regions of the array; and (b) observing a bindingpattern on the array and evaluating the presence of the targetpolynucleotide based on the observed binding pattern.